Inhibition of HIV Maturation via Selective Unfolding and Cross-Linking of Gag Polyprotein by a Mercaptobenzamide Acetylator.

For HIV to become infectious, any new virion produced from an infected cell must undergo a maturation process that involves the assembly of viral polyproteins Gag and Gag-Pol at the membrane surface. The self-assembly of these viral proteins drives formation of a new viral particle as well as the activation of HIV protease, which is needed to cleave the polyproteins so that the final core structure of the virus will properly form. Molecules that interfere with HIV maturation will prevent any new virions from infecting additional cells. In this manuscript, we characterize the unique mechanism by which a mercaptobenzamide thioester small molecule (SAMT-247) interferes with HIV maturation via a series of selective acetylations at highly conserved cysteine and lysine residues in Gag and Gag-Pol polyproteins. The results provide the first insights into how acetylation can be utilized to perturb the process of HIV maturation and reveal a new strategy to limit the infectivity of HIV.

Erythrocytes as carriers of antisense PNA addressed against HIV-1 gag-pol transframe domain.

PNA(PR2) is a peptide nucleic acid (PNA) complementary to a sequence of the viral protease-encoding gene, effective in blocking HIV release, when used at high doses. Erythrocytes (RBC) were used to target PNA(PR2) to the macrophage compartment. The antiviral activity was assessed in human HIV-infected macrophages both as inhibition of p24 production and reduction of HIV DNA content. PNA(PR2), either added to the medium at a concentration of 100 microM or loaded into RBC at about 40 microM, inhibited p24 production approximately 80% compared with infected samples and reduced HIV DNA content by 83% and 90%, respectively. The results show that (1) a stronger anti-HIV effect is achievable with higher doses of PNA(PR2), both when given free and encapsulated into RBC; (2) the antiviral effect obtained by free PNA(PR2) at a concentration of 100 microM is achievable by encapsulating it into RBC at a concentration of 40 microM, suggesting that RBC can be used as a delivery system to increase the antisense effect of PNA(PR2).

Loss of viral fitness associated with multiple Gag and Gag-Pol processing defects in human immunodeficiency virus type 1 variants selected for resistance to protease inhibitors in vivo.

We examined the viral replicative capacity and protease-mediated processing of Gag and Gag-Pol precursors of human immunodeficiency virus (HIV) variants selected for resistance to protease inhibitors. We compared recombinant viruses carrying plasma HIV RNA protease sequences obtained from five patients before protease inhibitor therapy and after virus escape from the treatment. Paired pretherapy-postresistance reconstructed viruses were evaluated for HIV infectivity in a quantitative single-cycle titration assay and in a lymphoid cell propagation assay. We found that all reconstructed resistant viruses had a reproducible decrease in their replicative capacity relative to their parental pretherapy counterparts. The extent of this loss of infectivity was pronounced for some viruses and more limited for others, irrespective of the inhibitor used and of the level of resistance. In resistant viruses, the efficiency of Gag and Gag-Pol precursor cleavage by the protease was impaired to different extents, as shown by the accumulation of several cleavage intermediates in purified particle preparations. We conclude that protease inhibitor-resistant HIV variants selected during therapy have an impaired replicative capacity related to multiple defects in the processing of Gag and Gag-Pol polyprotein precursors by the protease.